An antibody response capable of neutralizing not only homologous but also heterologous forms of the CXCR4-tropic human immunodeficiency virus type 1 (HIV-1) MNp and CCR5-tropic primary isolate HIV-1 JR-CSF was achieved through sequential immunization with a combination of synthetic peptides representing HIV-1 Env V3 sequences from field and laboratory HIV-1 clade B isolates. In contrast, repeated immunization with a single V3 peptide generated antibodies that neutralized only type-specific laboratory-adapted homologous viruses. To determine whether the cross-neutralization response could be attributed to a cross-reactive antibody in the immunized animals, we isolated a monoclonal antibody, C25, which neutralized the heterologous primary viruses of HIV-1 clade B. Furthermore, we generated a humanized monoclonal antibody, KD-247, by transferring the genes of the complementary determining region of C25 into genes of the human V region of the antibody. KD-247 bound with high affinity to the "PGR" motif within the HIV-1 Env V3 tip region, and, among the established reference antibodies, it most effectively neutralized primary HIV-1 field isolates possessing the matching neutralization sequence motif, suggesting its promise for clinical applications involving passive immunizations. These results demonstrate that sequential immunization with B-cell epitope peptides may contribute to a humoral immunebased HIV vaccine strategy. Indeed, they help lay the groundwork for the development of HIV-1 vaccine strategies that use sequential immunization with biologically relevant peptides to overcome difficulties associated with otherwise poorly immunogenic epitopes.

Although previous studies have shown that 50-200 antigen-major histocompatibility complex complexes (Ag-MHC) are sufficient to stimulate significant secretion of interleukin (IL)-2 from MHC class II-restricted T cell hybridomas, there have been no studies of this nature on more physiologically relevant T cell populations. In this study we have analyzed the ligand requirements for stimulation of responses from naive and previously primed T cells derived from T cell receptor (TCR)-transgenic animals whose TCR is specific for the pigeon cytochrome c (PCC) 88-104 peptide presented by I-Ek. Primed T cells were as sensitive as the previously reported T cell hybridomas, requiring about 100 Ag-MHC complexes to synthesize readily detectable quantities of IL-2, whereas naive T cells required 15 times more ligand to produce equivalent quantities of IL-2. Similarly, primed T cells required about 40 Ag-MHC complexes to produce a significant proliferative response, whereas naive T cells required about 400 complexes. In contrast to these results, naive and primed T cells showed similar ligand requirements when early events in the T cell activation pathway were analyzed; i.e. TCR down-modulation, CD69 and CD25 expression, and blast transformation. A further analysis of IL-2 and IL-2R expression indicated: 1) The first synthesis of IL-2 was detected at the same ligand concentration in both primed and naive T cells, but primed T cells made much more IL-2 as the ligand concentrations increased; 2) primed T cells expressed about fivefold more IL-2 receptor (R) than naive T cells, despite the fact that the antigen dose-response curves with respect to the percentage of cells expressing IL-2R were identical. These results suggest that naive and primed T cells have the same threshold with respect to the number of Ag-MHC complexes required to initiate T cell activation, but that due to the inefficient expression of IL-2 and IL-2R, engagement of more complexes is needed to enable naive T cells to synthesize the necessary amounts of these two molecules to allow T cells to go through a complete cycle of replication.

H-2K mice injected, intravenously in saline or intraperitoneally in incomplete Freund's adjuvant, with large quantities of the immunodominant I-Ek–restricted epitope from moth cytochrome c (MCC) 88–103 fail to respond to subsequent immunization with this epitope when administered in complete Freund's adjuvant. This state of tolerance can be broken by immunization with certain MCC 88–103 analogues that are heteroclitic antigens as assessed on representative MCC 88–103 specific T cell clones. In this paper, the mechanism of breaking tolerance by heteroclitic antigens was investigated. The following observations were made: (a) T cell hybridomas derived from tolerance-broken animals required higher concentrations of MCC 88–103 to be stimulated than hybridomas derived from normal immune animals, suggesting that they have T cell receptors (TCRs) of lower affinity; (b) in contrast to normal immune animals whose MCC-specific TCRs are typically Vβ3+/Vα11+, none of the hybridomas derived from tolerance-broken animals expressed Vβ3, although they were all Vα11+. Also, the Vβ complementarity determining region 3 (CDR3) regions from the tolerance-broken animals did not contain the canonical structure and length characteristics of the normal MCC 88–103 immune repertoire; and (c) adoptive transfer and tolerization of MCC-specific Vβ3+/Vα11+ transgenic T cells followed by immunization with heteroclitic antigen failed to terminate the state of tolerance. Collectively, these data strongly suggest that the mechanism involved in breaking tolerance in this system is the stimulation of nontolerized, low-affinity clones, rather than reversal of anergy. Further support for this mechanism was the finding that after activation, T cells apparently have a lowered threshold with respect to the affinity of interaction with antigen required for stimulation.

A thymic epithelial cell line transfected with I-E k was used in reaggregate cultures to study the role of peptides in positive selection of T cell receptor transgenic thymocytes. In this system, positive selection of CD4 SP cells occurred only after the addition of exogenous peptide. Analysis of antigen analogs indicated an inverse relationship between the antigenicity for peripheral T cells and the concentration of peptide required for positive selection. These data are most consistent with an avidity (rather than an affinity) model of positive selection, in which ligand density and the affinity of T cell receptor act in concert to determine the fate of developing thymocytes.

It has been previously established that effector and memory T cells are more sensitive to antigen stimulation than naive T cells. In this study, we compared the effect of ligand affinity on the activation of naive and effector T cells derived from pigeon cytochrome c (PCC)-specific TCR transgenic mice by stimulating these cells with a variety of ligands with widely differing antigenicity. The data obtained indicated the following. (i) The differences in antigen dose requirements for activation of naive and effector cells widened as the affinity of the antigen decreased. Most dramatically, peptides that were TCR antagonists for naive T cells were recognized as agonists by effector T cells. (ii) While both naive and effector T cells were activated by the bacterial superantigen staphylococcal enterotoxin A, specific for the transgenic TCR V(beta)3 chain, effector, but not naive, T cells were stimulated to proliferate by toxic shock syndrome toxin-1, a superantigen not previously described to be stimulatory for V(beta)3 T cells. (iii) Effector T cells, but not naive cells, proliferated in response to endogenous self-peptides presented by antigen-presenting cells in a syngeneic mixed lymphocyte reaction. Taken together these data indicate that effector T cells have a lower affinity threshold for activation than naive T cells. Further studies demonstrated that the heightened reactivity of effector T cells to low-affinity ligands declined progressively with repeated stimulations by antigen such that after repeated stimulation effector T cells were no longer stimulated by low-affinity ligands but recognized them as TCR antagonists similar to naive T cells.